Rhodotorula kratochvilovae CCY 20-2-26-The Source of Multifunctional Metabolites

microorganisms Article Rhodotorula kratochvilovae CCY 20-2-26—The Source of Multifunctional Metabolites Dana Byrtusová1,2 , Martin Szotkowski 2, Klára Kurowska 2, Volha Shapaval 1and Ivana Márová2,*   Citation: Byrtusová, D.; Szotkowski, M.; Kurowska, K.; Shapaval, V.; Márová, I. Rhodotorula kratochvilovae CCY 20-2-26—The Source of Multifunctional Metabolites. Microorganisms 2021,9, 1280. https://doi.org/10.3390/ microorganisms9061280 Academic Editor: Benedetta Turchetti Received: 29 April 2021 Accepted: 8 June 2021 Published: 11 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Faculty of Science and Technology, Norwegian University of Life Sciences, P.O. Box 5003, 1432 Ås, Norway; [email protected] (D.B.); [email protected] (V.S.) 2Faculty of Chemistry, Brno University of Technology, Purkyˇnova 464/118, 612 00 Brno, Czech Republic; [email protected] (M.S.); Winkler[email protected] (K.K.) *Correspondence: mar[email protected]; Tel.: +420-739-997-176 Abstract: Multifunctional biomass is able to provide more than one valuable product, and thus, it is attractive in the field of microbial biotechnology due to its economic feasibility. Carotenogenic yeasts are effective microbial factories for the biosynthesis of a broad spectrum of biomolecules that can be used in the food and feed industry and the pharmaceutical industry, as well as a source of biofuels. In the study, we examined the effect of different nitrogen sources, carbon sources and CN ratios on the co-production of intracellular lipids, carotenoids, β –glucans and extracellular glycolipids. Yeast strain R. kratochvilovae CCY 20-2-26 was identified as the best co-producer of lipids ( 66.7 ±1.5% of DCW), exoglycolipids (2.42 ± 0.08 g/L), β -glucan (11.33 ± 1.34% of DCW) and carotenoids ( 1.35 ±0.11 mg/g ), with a biomass content of 15.2 ± 0.8 g/L, by using the synthetic medium with potassium nitrate and mannose as a carbon source. It was shown that an increased C/N ratio positively affected the biomass yield and production of lipids and β-glucans. Keywords: Rhodotorula kratochvilovae; lipids; extracellular glycolipids; carotenoids; β-glucan 1. Introduction Multifunctional biomass, i.e., the possibility of isolation of a broad spectrum of metabolites from one single fermentation process, is the perspective task that establishes a costeffective microbial production and ensures the economic feasibility of microorganism cultures. The increasing demand for bio-based compounds has resulted in intensive screening of potential co-production candidates [ 1 , 2 ]. However, it is not easy to achieve high yields of all desired metabolites and cell biomass, since they require different culture strategies, and therefore, it is challenging to develop a balance between the medium composition and the suitable biomass and production of desired compounds. The co-production strategy focuses mostly on two-component yields, such as exopolysaccharides and lipids [ 2 ], carotenoids and lipids biosynthesis [3] or carotenoids and enzymes [4], of yeast. Basidiomycota, the phylum of the fungus kingdom, contains 30,000 described species, which includes mushroom-forming fungi, jelly fungi, yeast, rusts and smuts [ 5 ]. Among the mentioned phylum, there is carotenoids-producing yeast (called “carotenogenic” or “red” yeast), in which the genus of Rhodotorula is the most studied. This yeast can utilize a wide range of substrates, monoand polysaccharides and waste materials while producing highvalue substances [ 6 ]. Besides carotenoids, these unicellular microorganisms are capable of producing a variety of biotechnologically important metabolites, such as lipids [ 7 ], ergosterol [8], enzymes [9], extracellular molecules [10] and polysaccharides [11]. Carotenoids, the terpenoid pigments of 40 carbon atoms derived from two units of geranyl-geranyl transferase pyrophosphate, represent important bioactive molecules for human health [ 12 ]. They are lipid-soluble natural colorants, with a yellow to red color, that exhibit antioxidant activity and promote healthy effects, such as a reduction of the risk of degenerative diseases (heart diseases, cancer, cataract or macular degeneration) [ 13 ] Microorganisms 2021,9, 1280. https://doi.org/10.3390/microorganisms9061280 https://www.mdpi.com/journal/microorganisms Microorganisms 2021,9, 1280 2 of 18 and improving immunity [ 14 ], and they are also vitamin A precursors. Most of the carotenoids are extracted from vegetables, requiring seasonal and geographic variability, or are produced by chemical synthesis [ 12 ], generating hazardous wastes and by-products. Commercial microbial sources focus mainly on algal sources, such as Dunaliella species (producer of β -carotene), Chlorella (luteine) or Haematococcus (astaxanthin) [ 15 ]. On the other hand, carotenogenic yeast, mainly Rhodotorula genera, can synthesize specific carotenoids (thorulene, torularhodin, β -carotene) in different proportions and yields [ 16 ]. Currently, renewable energy has been gaining scientific interest due to increasing demands of fossil fuels and the rise of global warming and environmental pollution. Microbial systems, mainly carotenogenic oleaginous yeast, are efficient producers of triacylglycerols (TAGs) and are able to accumulate up to 70% of dry cell weight (DCW) together with high amount of biomass [ 17 ]. Additionally, after the extraction of lipophilic compounds from biomass, other valuable products can be obtained. Beta-glucans are a group of polysaccharides, consisting of glucose monomers connected with a β -glycosidic bond. They can be found in cell walls of high fungi (mushrooms), yeast, plants, algae and bacteria, where they are responsible for the mechanical and structural properties [ 18 , 19 ]. Their increasing market value is caused by its properties, such as immunostimulatory, antimicrobial, antitumor, anti-oxidative and anti-inflammatory activities [ 20 ]. For carotenogenic yeast, only a few papers can be found concerning β -glucans production [ 11 , 21 ]; thus, it is an attractive task for identifying new potential biotechnological candidates. The aim of this study was to assess the optimization of the production of a wide range of metabolites, from pigments, ergosterol and lipids to polysaccharides in the carotenogenic yeast Rhodotorula kratochvilovae CCY 20-2-26. The main goal of the presented study is to optimize the co-producing strategy and find an well-balanced and cheap culture medium. 2. Materials and Methods 2.1. Experimental Design Two biological replicates were prepared for each strain and cultivation medium by inoculating strains from frozen cryopreserved stocks on agar plates. Every biological replicate was prepared in a separate Erlenmeyer flask. At the diverse time points of cultivation, cell biomass was examined by gas chromatography for lipid analysis, liquid chromatography for carotenoids and ergosterol production and glucan analysis by the Yeast and Mushroom β -glucan Assay Procedure. The extracellular component was extracted by ethyl acetate. The experimental part starts with optimizing the culture conditions to enhance the biomass and multivariate metabolite production (nitrogen sources, carbon sources and the C/N ratios). 2.2. Yeast Strain and Media for Optimizing the Culture Conditions Carotenogenic Basidiomycetes yeast strain Rhodotorula kratochvilovae CCY 20-2-26 was obtained from the Culture Collection of Yeasts (Institute of Chemistry, Slovak Academy of Science, Bratislava, Slovakia). Cultivation of yeast was performed, first, on YPD agar medium (yeast extract, 10.0 g/L; peptone, 20.0 g/L; glucose 20.0 g/L; agar, 20.0 g/L) (Merck, Darmstadt, Germany) inoculated from frozen cryopreserved stock cultures and incubated for 72 h at 25 ◦ C. Inoculum was prepared by transferring 1 µ L of yeasts cells from YPD agar into 50 mL of sterile YPD broth medium (yeast extract, 10.0 g/L; peptone, 20.0 g/L; glucose 20.0 g/L) (Merck, Darmstadt, Germany) in an Erlenmeyer flask (250 mL) and cultivated for 24 h at 25 ◦ C under shaking regime (110 rpm, 50 mm). The cells were washed with sterile water and resuspended in fresh production medium in a volume ratio of 1:5 of YPD inoculum to production media (Tables 1and 2). The cultivation in production media was performed in Erlenmeyer flasks at 25 ◦ C under the constant shaking regime (110 rpm, 50 mm) and the samples were taken every 48, 72, 96 and 144 h. All cultivation media were sterilized at 121 ◦C for 15 min. Microorganisms 2021,9, 1280 3 of 18 Table 1. Composition of the culture media with different nitrogen sources (C/N ratio 70:1). Component g/L Glucose 40.42 KH2PO44 MgSO4·7H2O 0.7 Nitrogen sources g/L Yeast extract 2 KNO31.52 NH4Cl 0.80 (NH2)2SO40.9906 Urea 0.45 Table 2. Composition of the culture media with different carbon sources (C/N ratio 70:1). Component 1 [g/L] Component 2 [g/L] Urea—0.45 KNO3—1.52 KH2PO4—4 KH2PO4—4 MgSO4·7H2O—0.7 MgSO4·7H2O—0.7 Carbon sources g/L Mannose 40.42 Lactose 34.94 Glycerol 37.57 Xylose 36.75 Glucose 40.42 2.3. Preparation of Yeast Biomass for the Glucan, Lipid and Carotenoids Analysis After cultivation, the yeast biomass was centrifuged at 4500 rpm for 5 min at 4 ◦ C and the biomass pellet was then washed three times using 0.1% NaCl solution. Furthermore, yeast biomass was freeze-dried for 48 h and, subsequently, stored at −20 ◦C until use. 2.4. Analysis of Glucans by Yeast and Mushroom β-glucan Assay Kit The total glucan content and the content of β - and α -glucans were determined according to the Yeast and Mushroom β -glucan Assay Procedure K-YBGL (Megazyme/NEOGEN, Lansing, MI, USA) [ 22 , 23 ]. To estimate the total glucan content, freeze-dried yeast biomass was hydrolyzed with ice-cold 12 M sulfuric acid for 2 h and then incubated for 2 h at 100 ◦ C. Furthermore, acidic hydrolysate was neutralized with 200 mM sodium acetate buffer (pH 5) and 10 M KOH, followed by the effect of enzymes exoβ -(1 → 3)-D-glucanase and β -glucosidase in acetate buffer (pH 4.5) for 1 h at 40 ◦ C. These enzymes are able to hydrolyze extremely resistant beta-glycosidic bounds in glucans. The α -glucan content was determined after enzymatic hydrolysis with amyloglucosidase and invertase, which hydrolyze alpha-glycosidic bonds. The β -glucans content was calculated according to the recommendation of the assay kit procedure as the difference between total glucan and α -glucan content. The absorbance values indicating the total glucans and α -glucans content were obtained spectrophotometrically at 510 nm after adding glucose oxidase/peroxidase reagent. 2.5. Total Lipid Content and Analysis of Fatty Acid Profile Into a 2 mL glass crimp vial, 10 ± 2 mg of freeze-dried yeast biomass was weighed together with 1.8 mL of transesterification solvent (15% (v/v) H 2 SO 4 in methanol (HPLC grade), 0.5 mg/mL C17 as an internal standard) and the sample was incubated for 2 h at 85 ◦ C. After cooling to room temperature, the full volume of vial was transferred into a 4 mL glass vial with the addition of 0.5 mL 0.05 M NaOH and 1 mL of hexane (GC grade). The mixture was vortexed for 5 min, and after the phase separation, 0.1 mL upper hexane extract and 0.9 mL of pure hexane was transferred into glass vials for further GC analysis. Total lipid content (wt% of total FAMEs of the dry weight) and the fatty acid Microorganisms 2021,9, 1280 4 of 18 profile were performed by Thermo Scientific TRACE ™ 1300 Gas Chromatograph equipped with a Zebron ZB-FAME column, 30 m × 0.25 mm × 0.20 µ m and flame ionization detector (FID). The temperature gradient is presented in the Table 3. In total, 1 µ L of the sample was injected in split mode with an inlet temperature of 260 ◦ C. FAME standard mixture (C4—C24; Sigma Aldrich/Merck, Darmstadt, Germany) dissolved in hexane was used for the identification of the FAMEs. Quantification was based on the C17:0 internal standard and relative response factors (RRF) calculated from five-point calibration curves of the individual FAMEs present in the standard mixture. Table 3. Temperature gradient. Retention Time (min) Gradient (◦C/min) Target Temperature (◦C) Hold (min) 0 - - - 1 0 80 1 5 15 140 0 21.7 3 190 0 25.5 25 260 1 25.5 STOP - - 2.6. Analysis of Carotenoids The method for the isolation and analysis of carotenoid pigments and ergosterol was adapted from Szotkowski et. al (2019) [ 24 ]. Briefly, 15 ± 3 mg of freeze-dried biomass was weighed into plastic extraction tubes and rehydrated by the addition of 1 mL of distilled water. The water was removed by centrifugation (10,000 rpm/5 min/10 ◦ C), and to the pellet, 300 ± 20 mg of acid-washed glass beads (250–500 µ m diameter, Roth, Germany) and 1 mL of methanol was added. The rupture of the biomass was performed by 10 min of vortexing on the bench-top vortex (2500 rpm). The content of the PP tube was transferred into a glass reaction tube by washing it with a 2000 µ L of chloroform and the glass tube was vortexed for another 10 min; then, 1 mL of distilled water was added for the phase separation. After centrifugation (3000 rpm/5 min/4 ◦ C), the separated bottom chlorophorm phase with extracted pigments and ergosterol was evaporated under nitrogen at 25 ◦ C, followed by the addition of 1 mL of a mixture of ethylacetate:acetonitrile (20:60). The ethylacetate:acetonitrile mixture, containing extracted pigments, was filtered through a syringe filter (0.45 µ m, PTFE membrane, 13 mm) and transferred into glass vials for further HPLC analysis. The mobile phases consist of mixture A (84% of acetonitrile, 2% of methanol and 14% 0.1 M Tris-HCl (pH = 8) and mixture B (68% of methanol and 32% of ethylacetate). The conditions of separation are presented in Table 4. The contents of individual pigments (betacarotene, lycopene, torulene, torularhodin) were calculated using calibration standards according to [ 24 ]. Thermo Finnigan Surveyor HPLC/PDA system (Thermo Fisher Scientific, Waltham, MA, USA) and Xcalibur software was used for chromatography data analysis. Beta-carotene and lycopene were purchased from Sigma Aldrich/Merck, Darmstadt, Germany, torularhodin and torulene from CaroteNature, Ltd., Münsingen Switzerland. Table 4. Conditions of HPLC separation of carotenoids, sterols and ubiquinone. Column Kinetex, EVO 150 ×4.6 mm, 2.6 µm; Phenomenex Volume of the sample 20 µL Elution Gradient 0–13 min: from 100% A to 100% B linearly 13–19 min: 100% B 19–20 min: from 100% B to 100% A linearly 20–25 min: 100% A PDA 285 (ergosterol), 435, 450 and 680 nm (carotenoids) Temperature 25 ◦C Time of analysis 25 min Microorganisms 2021,9, 1280 5 of 18 2.7. Isolation and Characterization of Extracellular Glycolipids Exoglycolipids were isolated from culture medium according to the procedure from Wang et al. (2019) [ 25 ]. After cultivation, 20 mL of culture broth was centrifuged ( 6500 rpm/5 min ) and the supernatant was separated and washed with ethylacetate in two steps (5 mL for each step). The exoglycolipids contained biomass was washed twice with 5 mL of ethylacetate and centrifuged (6500 rpm/5 min). All ethylacetate fractions were merged together, evaporated under nitrogen flow and lyophilized. The dry weight of the extract was weighed to estimate the grams of extracellular glycolipids per liter of culture medium. The final crude product was stored at 4 ◦C for further analysis. Purified exoglycolipids fraction was then analyzed for lipid and sugar content. The lipid content and composition were analyzed according the procedure in Section 2.7. For sugar content, 100 mg of crude product was put into a 10 mL glass vial with a silicone screw cap, together with 3 mL of 1% H 2 SO 4 and heated to 100 ◦ C for 2 h. After hydrolysis, sample was neutralized with 5% NaOH solution. The sample was filtered via a 0.4 µ m filter into a 1.8 mL screw cap vial. The prepared sample was analyzed on a DIonex UltiMate 3000 series HPLC with an RI detector on Luna Omega Sugar Column ( 200 mm ×2.6 µm×5.0 mm ) using isocratic elution 75:25 ACN:MiliQ water at 30 ◦C for 30 min according to [24]. 3. Results 3.1. Biomass and Biochemical Profile of R. kratochvilovae CCY 20-2-26 Growth on Diverse Nitrogen Sources First, the optimization of cultivation conditions and medium composition was tested using various nitrogen and carbon sources. In the starting experiment, R. kratochvilovae CCY 20-2-26 grew in the presence of various nitrogen sources, such as potassium nitrate, yeast extract, ammonium sulfate, ammonium chloride and urea (see Table 1). Here, glucose was used as a sole carbon source at a C/N ratio of 70:1. Figure 1shows the biomass production through time at different nitrogen sources. Potassium nitrate, yeast extract and urea appear to be the most effective for the growth of R. kratochvilovae CCY 20-2-26, where the yields reached a value of about 9.0 g/L of biomass. The highest biomass was obtained using potassium nitrate at the 144 h of cultivation, namely 9.5 g/L. On the contrary, there is only a slightly increase in biomass when ammonium sulfate and ammonium chloride were used. Therefore, these sources were no longer used in the following experiments. In general, the highest biomass production was recorded at 144 h, when the yeast reached the stationary phase. Changes of glucose concentration in medium with different N sources is also documented in Table S1 (Supplementary File). The data confirmed that the highest degree of glucose utilization by R. kratochvilovae CCY 20-2-26 cells occurred in the presence of urea and potassium nitrate as N sources. These compounds were used in the following experiments, where various simple sugars were tested as carbon sources (see Section 3.2). The accumulation of metabolites was affected by changing the nitrogen sources. Potassium nitrate, yeast extract and urea showed a decrease of the beta-glucan yield through time. Conversely, the ammonium sulfate reached maximum values at the end of cultivation, 17.60 ± 1.84% w/w(Table 5). In Table S4 (Supplementary File) more detailed data are introduced regarding the production of alpha-glucans and beta-glucans. These data show that the production of alpha-glucans is very low and, in most of the cultivations, do not exceed 1–2%. Thus, the total intracellular glucans can be considered as the betaglucans with a small portion of alpha-glucans. Microorganisms 2021,9, 1280 6 of 18 Figure 1. Biomass production by R. kratochvilovae CCY 20-2-26 cultivated in the presence of various nitrogen sources. Table 5. Biomass composition of R. kratochvilovae CCY 20-2-26 cultured on different nitrogen sources. Nitrogen Metabolite 48 h 72 h 96 h 144 h KNO3 Total lipids (% of DCW) 30.2 ±1.9 42.5 ±2.1 59.6 ±1.3 49.8 ±2.5 Total carotenoids (mg/g) 1.00 ±0.27 1.25 ±0.13 1.36 ±0.14 2.48 ±0.13 Extr. glycolipids (g/L) 0.62 ±0.02 1.73 ±0.09 1.51 ±0.20 1.83 ±0.07 Beta-glucan (% of DCW) 22.05 ±2.21 18.31 ±1.83 15.24 ±1.24 14.39 ±0.77 YE Total lipids (% of DCW) 37.0 ±1.0 43.8 ±1.8 58.8 ±1.5 56.2 ±1.2 Total carotenoids (mg/g) 1.19 ±0.11 1.08 ±0.05 1.25 ±0.03 1.16 ±0.09 Extr. glycolipids (g/L) 0.51 ±0.04 0.75 ±0.05 1.17 ±0.20 1.28 ±0.08 Beta-glucan (% of DCW) 24.99 ±1.69 21.43 ±1.57 16.70 ±1.33 12.62 ±1.11 SA Total lipids (% of DCW) 25.1 ±2.0 34.6 ±2.4 37.2 ±0.5 27.0 ±2.1 Total carotenoids (mg/g) 1.30 ±0.02 1.48 ±0.16 1.61 ±0.03 1.38 ±0.06 Extr. glycolipids (g/L) 0.12 ±0.01 0.05 ±0.02 0.08 ±0.03 0.07 ±0.02 Beta-glucan (% of DCW) 12.41 ±1.02 13.98 ±1.04 16.54 ±1.00 17.60 ±1.84 NH4Cl Total lipids (% of DCW) 30.5 ±2.4 43.6 ±2.1 41.3 ±1.2 38.8 ±3.0 Total carotenoids (mg/g) 1.34 ±0.10 1.54 ±0.09 1.52 ±0.16 1.52 ±0.04 Extr. glycolipids (g/L) 0.13 ±0.01 0.31 ±0.05 0.32 ±0.09 0.23 ±0.08 Beta-glucan (% of DCW) 14.80 ±0.81 15.71 ±0.66 13.33 ±0.85 11.30 ±0.75 Urea Total lipids (% of DCW) 31.3 ±1.1 42.2 ±2.0 56.8 ±1.2 51.7 ±3.1 Total carotenoids (mg/g) 0.97 ±0.01 1.04 ±0.00 1.14 ±0.46 2.83 ±0.21 Extr. glycolipids (g/L) 0.58 ±0.02 0.88 ±0.05 1.51 ±0.07 1.57 ±0.06 Beta-glucan (% of DCW) 20.94 ±1.25 20.15 ±1.30 14.67 ±0.59 16.93 ±1.13 Abbreviations: YE: yeast extract; SA: ammonium sulphate. Intracellular lipids increased to almost 60% of the DCW at 96th with potassium nitrate, followed with yeast extract and urea. The most abundant fatty acids were oleic acid (46.6%), palmitic acid (18.3%), linoleic acid (18.6%), stearic acid (4.5%), α -linolenic and myristic acid (3.1 and 2.1%). The production of individual fatty acids on different N sources can be found in a more detailed form in Figure S1 (Supplementary File). These data confirmed that nitrogen sources had only a marginal influence on the fatty acid composition in the intracellular lipids of R. kratochvilovae CCY 20-2-26. Microorganisms 2021,9, 1280 7 of 18 The biosynthesis of exoglycolipids showed the same trend as at intracellular lipids, where maximum values reached up to 1.8 g/L (Figure 2, Table 5). The lowest yield of extracellular lipids was observed at 72 and 144 h of cultivation using ammonium sulfate, i.e., only 0.1 g/L. The composition of the glycolipid showed 44% lipid content, composed mainly from oleic acid (47.7%), linoleic acid (22%), palmitic acid (11.8%), linolenic acid (10%) and a minority of stearic and capric acid (4.9 and 2.3%). The fatty acids from glycolipid showed a similar composition to intracellular lipid, but with a higher content of palmitic acid. Figure 2. The presence of “oil droples” (black arrows) on the surface of culture medium of the yeast strain Rhodotorula kratochvilovae CCY 20-2-26. The carotenoids content varies from 0.97–2.83 mg/g of biomass. Potassium nitrate and urea increased the carotenoids production to over 2 mg/g of DCW at the end of cultivation. The optimal time point for maximum accumulation of intracellular lipids is 96 h (Table 5), while prolonged growth (144 h) can be seen more suitable for pigments and extracellular glycolipids production. Detailed distributions of individual carotenoid pigments in media with different N sources are documented in Figure S2 (Supplementary File). There are only small differences in the ratio of the main produced pigments—beta-carotene and more oxidized torulene + torularhodine in cells grown in the presence of different N sources. 3.2. Biochemical Profile of R. kratochvilovae CCY 20-2-26 Growth on Diverse Carbon Sources From previous experiments, potassium nitrate and urea were chosen as the best nitrogen sources. In further experiments, the consumption of different sugars (lactose, mannose, glycerol, xylose and glucose) as C sources were compared during cultivation in the presence of and potassium nitrate (Figure 3). These data confirmed glucose as the most preferred substrate for R. kratochvilovae CCY 20-2-26. However, in the medium with potassium nitrate as an N source, mannose was utilized by very similar kinetics as glucose (Figure 3B). Because of the comparable growth and biomass production of yeast cells on mannose and urea (Tables S2 and S3) and based on recently published data [ 11 , 24 ], only mannose was used in further experiments and compared with other sugars. The reason is that all these tested sugars could be obtained by hydrolysis of some waste by-products as a rest material. These nitrogen sources are cheap and simple for media preparation; moreover, they showed the highest yields of biomass, intracellular lipids and mainly exoglycolipids (Figure 1, Table 5). High yields were also observed for the yeast extract, which is, however, a demanding source due to its price compared to the two selected above. Regarding metabolite production, mannose was found to be a very efficient carbon source. Biomass production showed values of about 6.0 g/L at the beginning of the experiment, and up to 10.0 g/L of biomass after 144 h of cultivation. Thus, urea and mannose appear to be the best sources of carbon and nitrogen for biomass production over time. In the previous experiment, using urea as a nitrogen source, a maximum yield of about 9.0 g/L was Microorganisms 2021,9, 1280 8 of 18 obtained, when the carbon source was glucose. Here, it can be observed that even better results (up to 10.0 g/L) were obtained with the same nitrogen and mannose source as the carbon source (Figure 4). Thus, it seems that mannose is an even better source of carbon than glucose for this experiment, although the difference is not significant. The worst source in the experiment was lactose, which is the least suitable source of carbon for yeast, probably due to the absence of lactase to break down disaccharide into usable monosaccharides. The biomass yield was maximally only about 1.0–1.5 g/L. Lactose was followed, also as a less suitable carbon source, by glycerol, which showed values slightly above 2.0 g/L of biomass, and these values were very similar throughout the whole time horizon of cultivation. Xylose appears very similar to glycerol, which showed slightly better results with 3.0 g/L of biomass. Corresponding data regarding utilization of individual carbon sources are shown in Tables S2 and S3 (Supplementary File). Microorganisms 2021, 9, x FOR PEER REVIEW 8 of 18 (Figure 3B). Because of the comparable growth and biomass production of yeast cells on mannose and urea (Tables S2, S3) and based on recently published data [11,24], only mannose was used in further experiments and compared with other sugars. The reason is that all these tested sugars could be obtained by hydrolysis of some waste by-products as a rest material. These nitrogen sources are cheap and simple for media preparation; moreover, they showed the highest yields of biomass, intracellular lipids and mainly exoglycolipids (Figure 1, Table 5). High yields were also observed for the yeast extract, which is, however, a demanding source due to its price compared to the two selected above. Regarding metabolite production, mannose was found to be a very efficient carbon source. Biomass production showed values of about 6.0 g/L at the beginning of the experiment, and up to 10.0 g/L of biomass after 144 hours of cultivation. Thus, urea and mannose appear to be the best sources of carbon and nitrogen for biomass production over time. In the previous experiment, using urea as a nitrogen source, a maximum yield of about 9.0 g/L was obtained, when the carbon source was glucose. Here, it can be observed that even better results (up to 10.0 g/L) were obtained with the same nitrogen and mannose source as the carbon source (Figure 4). Thus, it seems that mannose is an even better source of carbon than glucose for this experiment, although the difference is not significant. The worst source in the experiment was lactose, which is the least suitable source of carbon for yeast, probably due to the absence of lactase to break down disaccharide into usable monosaccharides. The biomass yield was maximally only about 1.0–1.5 g/L. Lactose was followed, also as a less suitable carbon source, by glycerol, which showed values slightly above 2.0 g/L of biomass, and these values were very similar throughout the whole time horizon of cultivation. Xylose appears very similar to glycerol, which showed slightly better results with 3.0 g/L of biomass. Corresponding data regarding utilization of individual carbon sources are shown in Tables S2 and S3 (Supplementary File). (A) (B) Figure 3. Consumption of different carbon sources by R. kratochvilovae CCY 20-2-26. (A) Changes of the concentration of individual sugars in the medium with urea as an N source. (B) Changes of 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 24 48 72 96 120 144 Sugar g/L hours Lactose Mannose Glycerol Xylose glucose 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 1 24 48 72 96 120 144 sugar g/L hours Lactose Mannose Glycerol Xylose Glucose Figure 3. Consumption of different carbon sources by R. kratochvilovae CCY 20-2-26. ( A ) Changes of the concentration of individual sugars in the medium with urea as an N source. ( B ) Changes of the concentration of individual sugars throughout the cultivation in the medium with potassium nitrate as an N source. Table 6shows the production of intracellular lipids, carotenoids, extracellular glycolipids and β -glucan in yeast biomass cultured at different saccharides and glycerol with a combination of urea. The biomass content for lactose was too low for glucan measurements. The urea with combination of mannose, glycerol and xylose decreases the β -glucan content when compared to previous experiment with glucose. With mannose as a carbon source, glucan yield dropped from 13.75 ± 0.99 to 10.54 ± 0.76, probably due to the accumulation of other metabolites. Conversely, cultivation on glycerol and xylose showed a Microorganisms 2021,9, 1280 9 of 18 slight increase in β -glucan production at the end of cultivation. In Table 6, values of the total intracellular glucans are introduced, while detailed production of alpha-glucans and beta-glucans in media with different C sources and urea as an N source is illustrated in Table S5 (Supplementary File). Figure 4. Biomass production with diverse carbon sources and with urea as nitrogen source. Abbreviations: Lakt: lactose; Man: mannose; Gly: glycerol; Xyl: xylose. Table 6. Biomass composition of R. kratochvilovae CCY 20-2-26 cultured on different carbon sources with urea. Carbon Metabolite 48 h 72 h 96 h 144 h Lactose Total lipids (% of DCW) 9.0 ±2.3 8.6 ±1.6 8.9 ±1.0 9.2 ±2.1 Total carotenoids (mg/g) 0.32 ±0.08 0.87 ±0.13 0.82 ±0.19 0.70 ±0.10 Extr. glycolipids (g/L) 0.10 ±0.01 0.01 ±0.00 0.03 ±0.02 0.03 ±0.01 Beta-glucan (% of DCW) - - - - Mannose Total lipids (% of DCW) 37.0 ±2.3 32.4 ±2.2 55.8 ±3.8 51.1 ±4.1 Total carotenoids (mg/g) 1.01 ±0.18 1.02 ±0.20 1.13 ±0.22 1.39 ±0.19 Extr. glycolipids (g/L) 0.51 ±0.02 0.61 ±0.05 1.06 ±0.08 1.42 ±0.10 Beta-glucan (% of DCW) 13.75 ±0.99 10.59 ±0.76 10.28 ±1.83 9.87 ±0.67 Glycerol Total lipids (% of DCW) 14.9 ±3.6 16.8 ±2.8 15.5 ±1.4 17.2 ±2.3 Total carotenoids (mg/g) 0.97 ±0.11 1.14 ±0.23 1.18 ±0.25 1.21 ±0.18 Extr. glycolipids (g/L) 0.14 ±0.00 0.09 ±0.01 0.23 ±0.02 0.10 ±0.03 Beta-glucan (% of DCW) 3.83 ±0.54 4.19 ±0.67 5.00 ±0.37 5.37 ±0.80 Xylose Total lipids (% of DCW) 15.6 ±0.1 15.7 ±2.5 19.2 ±0.9 20.3 ±2.4 Total carotenoids (mg/g) 0.97 ±0.22 1.47 ±0.32 1.13 ±0.21 1.52 ±0.30 Extr. glycolipids (g/L) 0.10 ±0.01 0.12 ±0.02 0.15 ±0.05 0.03 ±0.01 Beta-glucan (% of DCW) 2.96 ±0.39 8.28 ±1.13 7.59 ±0.91 7.04 ±0.76 The production of exoglycolipids, using different carbon sources, is analogical to the production of biomass—the most suitable carbon source is mannose (1.4 ± 0.10 g/L). The next suitable source appears to be glycerol, where the maximum yield was reached after 96 h of cultivation, slightly above 0.2 g/L of exoglycolipids. Other sources showed very low values compared to mannose, around 0.1 g/L. The concentration of exoglycolipids was slightly lower in this experiment when compared to the previous experiment. Thus, glucose in combination with urea appears to be a more suitable source of the production of exoglycolipids. Regarding intracellular lipids, mannose was confirmed to be the most suitable carbon source. However, not so high values were achieved as in the cultivation with potassium nitrate and glucose (Table 5). Using mannose, the maximum value was reached at 96 h of Microorganisms 2021,9, 1280 16 of 18 β -glucan production within the cultivation time can be explained by the accumulation of lipids, as described above. Carotenoids from biomass of the Rhodotorula genus can be classified as low (less than 0.1 mg/g), medium (0.1–0.5 mg/g) and high (more than 0.5 mg/g) [ 12 ]. The best results were achieved for urea and potassium nitrate with a combination of glucose, 2.83 ± 0.21 and 2.48 ± 0.13 mg/g of biomass, respectively. With other carbon sources, glycerol shows a presence of 2.72 ± 0.24 mg/g of total carotenoids, accompanied by 2.21 ± 0.16 mg/g mannose for a C/N ratio of 70:1. The composition of carotenoid pigments was relatively stable at most of the cultivation conditions, independently from the type of carbon and nitrogen source, respectively. Major fractions formed beta-carotene and a mixture of more oxidized torulenes (torulene and torularhodin). Both these derivatives have a similar biological effect and, thus, they were evaluated as a sum of the total carotenoids. The production of the total carotenoids exhibited no substantial changes also with an increased C/N ratio, while the distribution of pigments was different. An increased C/N ratio led to gradually decreased production of beta-carotene, accompanied by increased production of oxidized torulenes. Taken together, this study provides insight into the co-production of four metabolites with the application of cheap synthetic medium. The studied strain, R. kratochvilovae CCY 20-2-26, represents an interesting candidate to study the simultaneous production of extracellular glycolipids, intracellular oils, glucans and carotenoids. 5. Conclusions By optimizing the culture media, R. kratochvilovae CCY 20-2-26 was able to produce a high number of intracellular lipids, β -glucans and carotenoids, together with a high biomass yield. In addition, extracellular glycolipid, secreted into culture media, had a lipid content of 44% and was composed mainly of oleic acid (47.7%), linoleic acid (22%), palmitic acid (11.8%) and linolenic acid (10%). Using high C/N ratio with mannose as a carbon source and potassium nitrate as nitrogen source, 15.2 ± 0.8 g/L of yeast biomass was achieved, containing significant amount of intracellular lipids (66.7 ± 1.5% of DCW), exoglycolipids (2.42 ± 0.08 g/L), β -glucans (11.33 ± 1.34% of DCW) and carotenoids ( 1.35 ±0.11 mg/g ). Similar results were obtained with glucose as a carbon source. Conversely, lactose, xylose and glycerol declined the overall production. Potassium nitrate appeared to be an effective and cheap alternative nitrogen source compare to yeast extract. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/microorganisms9061280/s1, Table S1: R. kratochvilovae CCY 20-2-26 growing on various nitrogen sources with glucose as a C-source. Changes of concentration of glucose in the media with individual N-sources [g/L], Table S2: R. kratochvilovae CCY 20-2-26 growing on various carbon sources with urea as N-source. Changes of concentration of individual sugars in the media [g/L], Table S3: R. kratochvilovae CCY 20-2-26 growing on various carbon sources with potassium nitrate as N-source. Changes of concentration of individual sugars in the media [g/L], Table S4: Total, α - and β -glucan content in R. kratochvilovae cells grown in glucose medium with diverse nitrogen sources, Table S5: Total, α - and β -glucan content in R. kratochvilovae cells grown at diverse carbon sources with urea as nitrogen source, Table S6: Total, α - and β -glucan content in R. kratochvilovae cells grown at diverse carbon sources with potassium nitrate as nitrogen source, Figure S1: Fatty acid composition of lipids produced by R. kratochvilovae (glucose medium; different N sources; 96 h cultivation, C/N 70), Figure S2: Distribution of carotenoid pigments produced by R. kratochvilovae (glucose medium; different N sources; 96 h cultivation, C/N 70), Figure S3: Fatty acid composition of lipids produced by R. kratochvilovae on different C sources (urea; 96 h cultivation), Figure S4: Fatty acid composition of lipids produced by R. kratochvilovae on different C sources (KNO 3 ; 96 h cultivation), Figure S5: Carotenoid pigment composition produced by R. kratochvilovae on different C sources (urea; 96 h cultivation), Figure S6: Carotenoid pigment composition produced by R. kratochvilovae on different C sources (KNO 3 ; 96 h cultivation), Figure S7: Fatty acid composition of lipids produced by R. kratochvilovae (mannose medium; different C/N ratio; 96 h cultivation), Microorganisms 2021,9, 1280 17 of 18 Figure S8: Distribution of carotenoid pigments produced by R. kratochvilovae (mannose medium; different C/N ratio; 96 h cultivation). Author Contributions: Conceptualization, I.M., V.S., D.B. and M.S.; investigation, D.B., I.M., M.S., V.S. and K.K.; methodology, D.B., M.S. and K.K.; validation, D.B., M.S. and I.M.; writing—original draft preparation, D.B., M.S. and I.M.; writing—review and editing, I.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the project Lipofungi NFR-BIONÆR 268305 and the project Byprovalue Nr 301834 of the Research Council of Norway. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Publicly available datasets were analyzed in this study. Data of this study are partially available in a publicly accessible repository—PhD Thesis of Dana Byrtusova, Dr. This data can be found here: https://www.vutbr.cz/studenti/zav-prace/detail/129244. Conflicts of Interest: The authors declare no conflict of interest. References 1. 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